I. Field of the Invention
This invention relates to methods and compositions for increasing nodulation, nitrogen fixation, plant growth and productivity in legumes by inoculation of legumes with Rhizobium spp.
II. Description of the Prior Art
Biological nitrogen fixation is the consequence of a complex and unique symbiosis between Rhizobium bacteria and legume host plants. The first stage in this process is the formation of nodules which occurs by the penetration of the host root hairs by rhizobial bacteria, followed by the formation of a rhizobial infection thread which moves into the host plant's root cortex, after which the rhizobial bacteria are encased in specialized plant cells and then undergo rapid multiplication. Subsequently, the rhizobial bacteria become pleomorphic, their nuclear material degenerates and the resulting bacteroids develop the enzyme complexes, particularly nitrogenase, required for nitrogen fixation (Paul, E. A. and F. E. Clark, 1989, Soil Microbiology and Biochemistry. Academic Press Inc. San Diego. pp. 182-192). The environmental, nutritional and physiological conditions required for rhizobial cell growth and the successful establishment of efficient nitrogen-fixing symbioses are known (Trinick, M. J., 1982, IN W. J. Broughton (Ed.), Nitrogen Fixation Vol. 2, Clarendon Press, Oxford. pp. 76-146).
The amounts of nitrogen fixed by legume:Rhizobium symbioses are significant and, in agricultural situations, can be used to supplement or replace nitrogen fertilizer applications. For example, a typical rate of nitrogen fixation by nodulated alfalfa is up to 250 kg/hectare/year (Atlas, R. M. and R. Bartha, 1981, Microbial Ecology: Fundamentals and Applications, Addison-Wesley Pub. Co. Reading. pp. 364-365) and up to 450 kg/ha/yr by nodulated soybeans (Peoples, M. B. and E. T. Craswell, 1992, Plant Soil 141: 13-39). Consequently, legume crops have become an integral component of most field crop rotations used in agriculture around the world.
Commercial inoculant compositions are commonly used when planting legume crops to ensure that sufficient rhizobial bacteria are present to establish effective nitrogen-fixing systems. Various types of commercial Rhizobium inoculant carriers, compositions and preparations are known including liquids, powders and granules (Thompson, J. A., 1991, IN Report of the Expert Consulation on Legume Inoculant Production and Quality Control (J. A. Thompson, Ed.) Food and Agriculture Association of the United Nations, Rome, pp. 15-32).
It appears that peat is the carrier of choice for the rhizobial inoculant industry (van Elsas, J. D. and C. E. Heijnen, 1990, Biol. Fertil. Soils 10: 127-133). Peat carriers may or may not be sterilized prior to inoculation with rhizobial cells. However, major concerns about the use of non-sterilized peat carriers include potential dissemination of human, animal or plant pathogens (Catroux, G. and No Amarger, 1992, IN Release of Genetically Engineered & Other Micro-organisms (J. C. Fry and M. J. Day) Cambridge University Press, Cambridge. pp. 1-13). It is known that the highest quality, the most stable and most efficacious rhizobial inoculants are manufactured in sterile carriers and are microbially pure (Day, J. M., 1991, IN Report of the Expert Consulation on Legume Inoculant Production and Quality control (J.A. Thompson, Ed.) Food and Agriculture Association of the United Nations, Rome, pp. 75-85). For these reasons, numerous international regulatory agencies are imposing strict limits on the presence, or the number, of contaminating microorganisms that can be tolerated in commercial rhizobial inoculants (Day, J. M., 1991, IN Report of the Expert Consulation on Legume Inoculant Production and Quality Control (J.A. Thompson, Ed.) Food and Agriculture Association of the United Nations, Rome, pp. 75-85).
Rhizobial titers in contaminated peat-based inoculants are significantly lower than the rhizobial titers in presterilized peat (Date, R. A. and R. J. Roughley, 1977, IN A Treatise on Dinitrogen Fixation (R. W. F. Hardy and A. H. Gibson (Eds.) John Wiley & Sons, New York, pp. 243-275). The reduction of rhizobial titre in unsterilized peat is sometimes due to the presence of contaminating microorganisms which inhibit the growth, or are otherwise antagonistic to the desired rhizobia (Olsen, P. E., W. A. Rice and M. M. Colins submitted to Soil Biol. Biochem. for publication). In particular, fungal contamination of peat can be a primary factor in reducing the quality and stability of rhizobial inoculants, and the impairing of their nodulation performance (Day, J. M., 1991, IN Report of the Expert Consulation on Legume Inoculant Production and Quality Control (J. A. Thompson, Ed.) Food and Agriculture Association of the United Nations, Rome, pp. 75-85; Sparrow, Jr., S. D. and G. E. Ham, 1983, Agron. J. 75:181-184). Consequently, some commercial rhizobial inoculant production processes have the objectives of eliminating all microbial contamination, including the presence of fungal organisms, from the inoculant carrier substrates prior to inoculation with rhizobial cultures, and then maintaining microbial purity until these products are used to inoculate legume seeds just prior to planting (Day, J. M., 1991, IN Report of the Expert Consulation on Legume Inoculant Production and Quality Control (J. A. Thompson, Ed.) Food and Agriculture Association of the United Nations, Rome, pp. 75-85).
It is known that optimal rhizobial culture performance, and optimal nodulation and nitrogen-fixation processes in legume:Rhizobium symbioses require significant energy expenditures and benefit considerably from supplemental phosphate inputs (Beck, D. P. and D. N. Munns, 1984, Appl. Environ. Microbiol. 47:278-282; Israel, D. W., 1987, Plant Physiol. 84: 835-840). Optimizing the phosphate nutrition of legume crops, i.e. increasing the availability of soluble phosphate for plant uptake, will maximize nitrogen fixation and productivity of legume crops (Adu-Gyamfi, J. J., K. Fujita and S. Ogata, 1989, Plant Soil 119: 315-324; Griffeth, W. K., 1986, IN Phosphorus for Agriculture; A Situation Analysis. Potash & Phosphate Institute, Atlanta. pp. 57-62; Keyser, H. H. and F. Li., 1992, Plant Soil 141: 119-135).
It is known that soil microbial populations include a wide variety of bacterial and fungal species, each of which specifically interact with and affect the growth of all the other species by competing for nutrients and through the production of stimulatory or inhibitory compounds (Curl, E. A. and B. Truelove, 1986, The Rhizophere, Springer-Verlag, pp. 140-141). Generally, two types of interactions occur between soil microorganisms, i.e., growth promotion or growth inhibition. It is clear that these types of interactions are most intense in habitats which contain high levels of nutrients, such as root regions.
It is known that some soil bacteria, primarily Gramnegative strains, are capable of stimulating nodulation by rhizobial bacteria, and are referred to as "nodulation-promoting rhizobacteria" (Polonenko, D. R., J. W. Kloepper and F. M. Scher, European patent application Serial No. 863093498 published July 1st, 1987 under publication no. 0 227 336 A1). However, the mechanisms by which nodulation-promoting rhizobacteria stimulate nodulation have not been discovered.
It is also known that a variety of soil microorganisms are capable of increasing the availability of phosphate for plant uptake (Kucey, R. M. N., H. H. Janzen and M. E. Leggett, 1989, Adv. Agron. 42: 199-228). Several studies have assessed the beneficial effects of "phospho-bacteria" on nodulation and nitrogen fixation in beans (Grimes, H. D. and M. S. Mount, 1984, Soil Biol. Biochem. 16: 27-30) and chickpea (Alagawadi, A. R. and A. C. Gaur, 1988, Plant Soil 105:241-246). In these studies, however, it has not been determined whether the benefits are due to increased phosphate availability or to microbial production of plant growth hormones (Alagawadi, A. R. and A. C. Gaur, 1988, Plant Soil 105:241-246; Grimes, H. D. and M.S. Mount, 1984, Soil Biol. Biochem. 16: 27-30). Furthermore, Badr El-Din et. al. (Badr El-Din, S. M. S., M. A. Khalafallah, and H. Moawad, 1986, Z. Pflanzenernaehr. Bodenk. 149:130-135) found that dual inoculation of "phospho-bacteria" and Rhizobium japonicum (also known as Bradyrhizobium japonicum) had no effect on nodule dry weight or nitrogen uptake of soybeans in a field experiment.
A unique sub-group of soil fungi which establish symbiotic relationships with plants by penetrating the roots and then forming specialized fungal structures within the host plant root systems, i.e. vesicular-arbuscular mycorrhizae (VAM), can also significantly improve nodulation and nitrogen fixation by increasing phosphate availability to mycorrhizal legumes (Barea, J. M. and C. Azcon-Aguilar, 1983, Adv. Agron. 36:1-54). VAM absorb soluble phosphate from the soil solution into their mycelia, and then translocate the phosphate to within the plant roots (Harley, J. L. and S. E. Smith, 1983, Mycorrhizal Symbiosis. Academic Press, London. pp. 78-84). VAM do not dissolve solid native or precipitated phosphates (Harley, J. L. and S. E. Smith, 1983, Mycorrhizal Symbiosis. Academic Press, London. pp. 84-86).
On the other hand, rhizobial bacteria are weakly competitive within soil microbial populations and, consequently, tend to be negatively affected by the presence of antibiotic-producing microorganisms. It appears that the most common antibiotic-synthesizing microorganisms are most abundant in the root regions of cultivated plants, and generally are species of Penicillium, Streptomyces, Trichoderma, Aspergillus, Bacillus and Pseudomonas (Curl, E. A. and B. Truelove, 1986, The Rhizophere, Springer-Verlag, p. 153-154). A study with a forage legume demonstrated that certain antibiotic-producing fungi species, particularly in the genus Penicillium, inhibited the activity of rhizobial bacteria and the formation of nodules (Holland, A. A. and C. A. Parker, 1966, Plant Soil 25: 329-340). Furthermore, a study with a grain legume found that a Penicillium sp. significantly reduced nitrogen fixation (Downey, J. and C. van Kessel, 1990, Biol. Fertil. Soils 10:194-196). They hypothesized that the Penicillium produced organic acids which acidified the rhizosphere and thus interfered with the rhizobial performance.
It has been clearly demonstrated that the soil fungus Penicillium bilaii increases the availability of soluble phosphate for plant uptake by dissolving solid forms of phosphate (Kucey, R. M. N., U.S. Pat. No. 5,026,417 issued Jun. 25, 1991). P. bilaii does not penetrate into plant roots but rather, colonizes root surfaces and the immediate rhizosphere. This organism is registered under the Canadian Fertilizers Act (Reg. Nos. 900025A, 920064A) as a fertilizer supplement for use to increase phosphate availability to wheat, canola, pea and lentil. Kucey (Kucey, R., 1987, Appl. Environ. Microbiol. 53:2699-2703) found that dual-inoculation of field beans with P.bilaii and R.phaseoli did not have statistically significant effects on phosphate uptake or bean plant dry weights, when grown in autoclaved soil in a greenhouse trial.
Moreover, Downey and van Kessel (Downey, J. and C. van Kessel, 1990, Biol. Fertil. Soils 10:90-194-196) demonstrated that in greenhouse trials, nitrogen fixation and assimilation were significantly reduced in pea plants that were "dual-inoculated" with P. bilaii and Rhizobium leguminosarum bv. viceae when compared to peas inoculated with R. leguminsarum bv. viceae alone.